EP1806401B1 - Dna encoding novel enzyme having d-serine synthase activity, method of producing the enzyme and method of producing d-serine by using the same - Google Patents

Dna encoding novel enzyme having d-serine synthase activity, method of producing the enzyme and method of producing d-serine by using the same Download PDF

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EP1806401B1
EP1806401B1 EP05793824.3A EP05793824A EP1806401B1 EP 1806401 B1 EP1806401 B1 EP 1806401B1 EP 05793824 A EP05793824 A EP 05793824A EP 1806401 B1 EP1806401 B1 EP 1806401B1
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serine
transformant
treatment
formaldehyde
protein
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EP1806401A1 (en
EP1806401A4 (en
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Tadashi Araki
Tomonori Hidesaki
Seiichi Watanabe
Keita Nishida
Kiyoteru Nagahara
Mitsuo Koito
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Mitsui Chemicals Inc
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Mitsui Chemicals Inc
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Priority to EP13154224.3A priority patent/EP2602320B1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1014—Hydroxymethyl-, formyl-transferases (2.1.2)

Definitions

  • the present invention relates to DNA encoding a novel enzyme having activity of synthesizing D-serine from formaldehyde and glycine, recombinant DNA constructed by integrating such DNA into a vector, a transformant transformed with the recombinant DNA, a novel enzyme having activity of synthesizing D-serine from formaldehyde and glycine, and a method for producing D-serine from formaldehyde and glycine with the use of the enzyme.
  • D-serine has been known as a compound that is useful as a synthesis intermediate for medicaments such as D-cycloserine.
  • DTA D-threonine aldolase
  • DTA derived from Arthrobacter sp. DK-38 which is a microorganism of the same genus Arthrobacter, has a broad substrate specificity that responds to D-threonine, D- ⁇ -hydroxyphenylserine, D- ⁇ -hydroxy- ⁇ -aminovaleric acid, and the like; however, it does not react with D-serine (see Eur. J. Biochem., 1997, 248, pp. 385-393 ).
  • D- ⁇ -hydroxyamino acids can be produced from glycine and an aldehyde compound with the use of DTA derived from the genus Xanthomonas ( JP Patent Publication (Kokai) No. 5-168484 A (1993 )).
  • D-serine can be synthesized from formaldehyde and glycine with the use of DTA derived from the genus Xanthomonas.
  • the present inventors thought that some unknown enzymes that have structures similar to those of known DTAs could be capable of synthesizing D-serine from formaldehyde and glycine. Thus, they examined information regarding the amino acid sequences of known DTAs. Accordingly, they have found information regarding the amino acid sequences of DTAs derived from the genera Xanthomonas (GenBank accession No. E05055), Achromobacter (GenBank accession No. AB026892), and Arthrobacter (GenBank accession No. AB010956). As a result of comparison and examination of these amino acid sequences, they have found that amino acid sequences corresponding to the N-terminal region and the C-terminal region of DTA are highly homologous to each other.
  • the present inventors designed primers based on these amino acid sequences of the N-terminal region and the C-terminal region. They then tried to amplify chromosome DNAs of a variety of microorganisms by PCR using the primers. Accordingly, they have succeeded in amplifying DNA encoding an enzyme having activity of synthesizing D-serine from formaldehyde and glycine with the use of a microorganism of the genus Achromobacter.
  • the amino acid sequence corresponding to the DNA is approximately 50% homologous to the amino acid sequence of known DTA derived from the genus Achromobacter, such that it significantly differs from the amino acid sequence of known DTA derived from the genus Achromobacter. Meanwhile, it is approximately 90% homologous to the amino acid sequence of known DTA derived from the genus Xanthomonas.
  • D-serine was synthesized by allowing formaldehyde to react with glycine using recombinant Escherichia coli that had been transformed with a recombinant DNA constructed by integrating the above novel DNA into a vector.
  • DTA unlike the case of conventionally known DTA, they have found that D-serine can be synthesized with a reaction yield of as high as 70% or more from 100 mM glycine and 100 mM formaldehyde.
  • L-serine be mixed in D-serine. Formation of L-serine as a byproduct upon production of D-serine causes a significantly lowered purification yield of D-serine, since the solubility of DL-serine is lower than that of D-serine.
  • L-serine as a byproduct can be restrained by carrying out organic solvent treatment and/or heat treatment in the presence of divalent metal ions.
  • formation of L-serine as a byproduct can be restrained by maintaining formaldehyde concentration at 150 mM or more during reaction.
  • formation of L-serine as a byproduct can be restrained by using a microorganism lacking an L-serine synthase gene as a host producing DSA that is used for D-serine synthesis.
  • the present invention provides a protein having enzyme activity of synthesizing D-serine from glyine and formaldehyde, DNA encoding such proteins, recombinant DNA constructed by integrating such DNA into a vector, non-human transformants, enzyme production methods, and D-serine production methods, all as defined in the accompanying claims.
  • DNA comprising the amino acid sequence set forth in SEQ ID NO: 4, 6, or 8 in the Sequence Listing and encoding an enzyme having activity of synthesizing D-serine from glycine and formaldehyde (hereafter to be abbreviated as "DSA") can be obtained by extracting chromosomal DNA from, for example, Achromobacter xylosoxidans (ATCC9220), which is available from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A.), and Achromobacter xylosoxidans (NBRC13495) and Achromobacter denitrificans (Synonym: Achromobacter xylosoxidans subsp.
  • Achromobacter xylosoxidans ATCC9220
  • Achromobacter xylosoxidans NBRC13495
  • Achromobacter denitrificans Sesp.
  • denitrifcans (NBRC15125), which are available from the NITE Biological Resource Center of the National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan) and carrying out PCR with the use of primers comprising the nucleotide sequences set forth in SEQ ID NOS: 1 and 2 in the Sequence Listing, respectively.
  • a gene that is amplified with difficulty can be occasionally amplified by a method wherein the annealing temperature is controlled at a low temperature, by a method involving the addition of dimethyl sulfoxide (approximately 5%) to a PCR reaction solution, by a method using a PCR kit (e.g., GC-RICH PCR System produced by Roche) that is designed to readily amplify a GC-rich gene, or by a combination of the above methods or the like.
  • a PCR kit e.g., GC-RICH PCR System produced by Roche
  • DNA encoding DSA include the DNA nucleotide sequence of a DTA gene derived from Achromobacter xylosoxidans (ATCC9220) set forth in SEQ ID NO: 3, the amino acid sequence (translated by such nucleotide sequence) set forth in SEQ ID NO: 4, the DNA nucleotide sequence of a DTA gene derived from Achromobacter xylosoxidans (NBRC13495) set forth in SEQ ID NO: 5, the amino acid sequence (translated by such nucleotide sequence) set forth in SEQ ID NO: 6, the DNA nucleotide sequence of a DTA gene derived from Achromobacter denitrificans (NBRC15125) set forth in SEQ ID NO: 7, and the amino acid sequence (translated by such nucleotide sequence) set forth in SEQ ID NO: 8.
  • DNA encoding a protein having DSA activity and comprising an amino acid sequence derived from the amino acid sequence set forth in SEQ ID NO: 4, 6, or 8 by deletion, substitution, insertion, or addition of one to several amino acid residues via introduction of adequate mutation such as deletion, substitution, insertion, and/or addition with the use of site-specific mutagenesis methods ( Nucleic Acid Res., 10, p. 6487 (1982 ); Nucleic Acid Res., 13, p. 4431 (1985 ); Methods in Enzymol., 100, p. 448 (1983 ); Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989 ); PCR A Practical Approach IRL Press p.
  • amino acid residues described herein can be carried out by the aforementioned site-specific mutagenesis methods that were known techniques prior to the filing of the present application.
  • the term "one to several amino acid residues” indicates a number of amino acids that allows deletion, substitution, insertion, or addition of amino acid residues (namely, 1 to 5 amino acid residues and preferably 1 to 3 amino acid residues) to be carried out by site-specific mutagenesis methods.
  • conditions for hybridization include conditions that are generally used by persons skilled in the art to detect particular hybridization signals.
  • such conditions indicate stringent hybridization conditions and stringent washing conditions.
  • such conditions involve overnight incubation at 55°C with the use of a probe in a solution containing 6 ⁇ SSC (1 ⁇ SSC composition: 0.15 M NaCl and 0.015 M sodium citrate (pH 7.0)), 0.5% SDS, 5 ⁇ Denhardt's solution, and 100 mg/ml herring sperm DNA.
  • Such conditions also involve washing of a filter in 0.2 ⁇ SSC at 42°C.
  • Stringent conditions involve a use of 0.1 ⁇ SSC at 50°C during a step of washing a filter.
  • a condition using 0.1 ⁇ SSC at 65°C during the same step can be explained.
  • a recombinant DNA can be obtained by integrating the aforementioned DNA encoding DSA into a vector.
  • a vector that is appropriate for cloning is a vector that is constructed for gene recombination with the use of a phage or plasmid that is capable of autonomously replicating in a host microorganism.
  • a phage or plasmid that is capable of autonomously replicating in a host microorganism.
  • examples of such phage include Lambda gt10 and Lambda gt11.
  • examples of such plasmid include pBTrp2, pBTac1, and pBTac2 (produced by Boehringer Mannheim), pKK233-2 (produced by Pharmacia), pSE280 (produced by Invitrogen), pGEMEX-l (produced by Promega), pQE-8 (produced by QIAGEN), pQE-30 (produced by QIAGEN), pBluescriptII SK+ and pBluescriptII SK(-) (produced by Stratagene), pET-3 (produced by Novagen), pUC18 (produced by Takara Shuzo Co., Ltd.), pSTV28 (produced by Takara Shuzo Co., Ltd.), pSTV29 (produced by Takara Shuzo Co., Ltd.), and pUC118 (produced by Takara Shuzo Co., Ltd.).
  • any promoter may be used as long as it can be expressed in a host cell.
  • promoters derived from Escherichia coli, phages, or the like such as a trp promoter (P trp ), a lac promoter (P lac ), a P L promoter, a P H promoter, and a P SE promoter.
  • promoters and the like that are artificially designed and modified such as a tac promoter and a lacT7 promoter, can be used.
  • Np promoter JP Patent Publication (Kokoku) No. 8-24586 B (1996 )
  • any sequence can be used as long as it can be expressed in a host cell.
  • a plasmid in which a Shine-Dalgarno sequence and an initiation codon are adjusted to have an adequate distance (e.g., 6 to 18 bases) therebetween.
  • a protein in which the N-terminal of a protein having activity of the protein or a protein derived from such a protein by deletion of a portion thereof is fused with the N-terminal of a protein encoded by an expression vector, may be expressed.
  • a transcription termination sequence is not always necessary for the expression of a protein of interest. However, it is preferable to place a transcription termination sequence directly below a structural gene.
  • a vector fragment by cleaving a vector as described above with a restriction enzyme used for cleavage of the aforementioned DNA encoding DSA.
  • the same restriction enzyme used for cleavage of the DNA is not necessarily used.
  • a method for binding the DNA fragment and a vector DNA fragment may be used as long as a known DNA ligase is used in such a method. For instance, after annealing of a cohesive end of the DNA fragment and that of a vector fragment, a recombinant vector of the DNA fragment and the vector DNA fragment is produced with the use of an adequate DNA ligase.
  • a non-human transformant transformed with the aforementioned recombinant DNA can be obtained by introducing the aforementioned recombinant DNA into a host cell.
  • a host cell there is no particular limitation, as long as a recombinant vector is stable and can autonomously replicate and a foreign gene is phenotypically expressed therein.
  • Examples of such host cell include microorganisms such as bacteria, including Escherichia coli such as Escherichia coli DH5 ⁇ and Escherichia coli XL-1 Blue.
  • microorganisms such as bacteria, including Escherichia coli such as Escherichia coli DH5 ⁇ and Escherichia coli XL-1 Blue.
  • yeasts or insect cells it is possible to use other microorganisms such as yeasts or insect cells as host cells.
  • the method for transferring recombinant DNA into the microorganism that can be used is a competent cell method using calcium treatment, an electroporation method, or the like.
  • a host cell for the purpose of restraining degradation of D-serine as a product, it is possible to use a microorganism having low D-serine deaminase activity or a microorganism lacking D-serine deaminase activity.
  • a microorganism lacking D-serine deaminase activity include Escherichia coli having a recombinant D-serine deaminase gene described in Example 6.
  • a microorganism having low activity of an enzyme involved in L-serine synthesis such as alanine racemase, serine hydroxymethyltransferase, or L-threonine aldolase, or a microorganism lacking such enzyme activity is preferably used so that L-serine is not produced.
  • a microorganism lacking all of the above enzymes is used as a host cell.
  • DSA can be produced by culturing the aforementioned transformant and collecting the thus obtained DSA.
  • a transformant can be cultured in accordance with a usual method used for culture of host cells.
  • a transformant is a procaryotic microorganism such as Escherichia coli or a eucaryotic microorganism such as a yeast
  • a medium in which such a microorganism is cultured may be a natural or synthetic medium as long as it contains carbon sources, nitrogen sources, inorganic salts, and the like, which cause assimilation of the microorganism, and as long as a transformant can efficiently be cultured therein.
  • carbon sources examples include: glucose, fructose, or sucrose; molasses containing any thereof; carbohydrates such as starch and starch hydrolysate; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol, as long as they can cause assimilation of a transformant.
  • nitrogen sources examples include: ammonia; a variety of ammonium salst of inorganic or organic acid, such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; other nitrogen-containing compounds; peptone; meat extracts; yeast extracts; corn steep liquor; casein hydrolysate; soybean cake; soybean cake hydrolysate; and a variety of fermentation bacteria and digests thereof.
  • inorganic salts examples include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
  • Culture is carried out under aerobic conditions used for shake culture, submerged cultivation under aeration and agitation, or the like.
  • Culture temperature is preferably 15°C to 50°C.
  • Culture time is 16 hours to 5 days, in general.
  • pH is maintained between 3.0 and 9.0. The pH is adjusted with the use of inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.
  • antibiotics such as ampicillin and tetracycline may be added to the medium according to need.
  • an inducer may be added to the medium according to need. For instance, when a microorganism transformed with an expression vector in which a lac promoter is used is cultured, isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) or the like may be added to the medium. Also, when a microorganism transformed with an expression vector in which a trp promoter is used is cultured, indoleacetic acid (IAA) or the like may be added to the medium.
  • IAA indoleacetic acid
  • a transformant can be separated and recovered by means of centrifugation, filtration, or the like, of a culture solution containing the transformant.
  • a treated product of a transformant can be obtained by allowing the transformant to be subjected to mechanical disruption, ultrasonication, freezing and thawing treatment, drying treatment, pressurization or depressurization treatment, osmotic pressure treatment, autodigestion, surfactant treatment, or enzyme treatment for the purpose of cell disruption. Also, a treated product of a transformant can be obtained as an immobilized fraction or transformant, which comprises DSA obtained via such treatment.
  • the treated product of a microorganism of the present invention is also referred to as a product subjected to a treatment similar to that used for the aforementioned treated product of a transformant.
  • transformed cells are disrupted and a cell disruption solution is subjected to a combination of, for example, fractionation via ion-exchange resin or gel-filtration chromatography or the like and salting out with the use of ammonium sulfate or the like, such that a purified enzyme can be obtained.
  • D-serine can be produced by allowing glycine to react with formaldehyde in the presence of the aforementioned transformant or a treated product of the transformant.
  • Production of D-serine is preferably carried out under conditions of shaking or agitation at pH 6.0 to 9.0 and at a temperature of 20°C to 60°C.
  • the amount of transformant or treated product of the transformant used is not particularly limited, as long as the reaction between formaldehyde and glycine progresses well.
  • a transformant or a treated product of the transformant is preferably added in an amount of at least 10 units and preferably 50 units or more in terms of DSA activity relative to 1 g of glycine.
  • such transformant may be added at once upon the initiation of reaction, at several different times, or continuously throughout reaction.
  • capacity for synthesizing 1 ⁇ mol of D-serine per 1 minute is defined as 1 unit.
  • DSA activity is calculated by measuring the amount of D-serine that is produced in a manner such that an enzyme solution is added to 200 mM Tris-hydrochloric acid buffer (pH 8.0) containing 100 mM glycine, 0.1 mM pyridoxal phosphate, 10 mM magnesium chloride, and 5 mM formaldehyde, followed by incubation at 30°C.
  • Glycine concentration in a reaction solution is 100 mM or more and preferably between 1 M and 5 M. In accordance with a method for adding glycine, it may be added at once upon the initiation of reaction, at several different times, or continuously along with the progress of reaction.
  • formaldehyde in the form of gas into a reaction solution. Also, it can be supplied in the form of an aqueous or alcohol solution.
  • formaldehyde paraformaldehyde can also be used as a supply source of formaldehyde.
  • an aqueous solution of approximately 37% formaldehyde is preferably used.
  • formaldehyde concentration in a reaction solution is controlled at a concentration that allows DSA activity not to be inhibited.
  • a concentration that allows DSA activity not to be inhibited is generally 5 M or less, preferably 2 M or less, further preferably 500 mM or less, and particularly preferably 300 mM or less.
  • formaldehyde can be added by the following methods for controlling formaldehyde concentration in a reaction solution: (1) a method for adding formaldehyde in a reaction solution at a given rate; (2) a method wherein formaldehyde concentration is quantified such that formaldehyde is added at several different times at a concentration that allows enzyme activity not to be deactivated; (3) a method for substantially avoiding inhibition due to formaldehyde, wherein paraformaldehyde is added and an enzyme is added to a reaction system at a rate that exceeds the rate at which formaldehyde is released from paraformaldehyde; and (4) a method for adding formaldehyde in an amount at which an increased pH level can be corrected.
  • D-serine as a product accumulates in a reaction solution along with the progress of reaction, the amount of glycine as a starting material decreases, and the pH of a reaction solution decreases along with the progress of the reaction.
  • the isoelectric point of D-serine is 5.68 and that of glycine is 5.97, such that alkali is added to the reaction solution in an amount that exceeds an amount necessary for correction of a decrease in the pH of the reaction solution, resulting in an increased pH level.
  • alkali metal hydroxide such as lithium hydroxide, sodium hydroxide, or potassium hydroxide
  • ammonium hydroxide calcium hydroxide
  • dipotassium phosphate disodium phosphate
  • potassium pyrophosphate sodium hydroxide
  • ammonia ammonia
  • a medium used for a reaction solution is water, an aqueous medium, an organic solvent, or a mixture solution of water or an aqueous medium and an organic solvent.
  • an aqueous medium that is used include buffer solutions such as a phosphate buffer solution, a HEPES (N-2-hydroxyethylpiperazin-N-ethanesulfonic acid) buffer solution, and a Tris (Tris(hydroxymethyl)aminomethane) hydrochloric acid buffer solution.
  • buffer solutions such as a phosphate buffer solution, a HEPES (N-2-hydroxyethylpiperazin-N-ethanesulfonic acid) buffer solution, and a Tris (Tris(hydroxymethyl)aminomethane) hydrochloric acid buffer solution.
  • an organic solvent that may be used include any organic solvent such as acetone, ethyl acetate, dimethyl sulfoxide, xylene, methanol, ethanol, or butanol, unless it inhibits
  • reaction yield may be improved with the addition of a compound having divalent metal ions, reductants such as 2-mercaptoethanol, dithiothreitol, and sodium bisulfite, and coenzymes such as pyridoxal phosphate and an ammonium salt.
  • reductants such as 2-mercaptoethanol, dithiothreitol, and sodium bisulfite
  • coenzymes such as pyridoxal phosphate and an ammonium salt.
  • Examples of a compound having divalent metal ions include magnesium chloride, manganese chloride, cobalt acetate, ferrous sulfate, and calcium chloride.
  • the concentration of such compound in a reaction solution is generally 0.1 mM to 100 mM and preferably 0.1 mM to 10 mM.
  • Formation of L-serine as a byproduct can be restrained by allowing glycine to react with formaldehyde in the presence of a treated product obtained by allowing the aforementioned transformant or a treated product of the transformant to be subjected to heat treatment or in the presence of a treated product obtained by allowing the aforementioned transformant or a treated product of the transformant to be subjected to organic solvent treatment.
  • any conditions may be applied, provided that thereby DSA activity is not significantly reduced by heating and L-serin production activity can be reduced or eliminated.
  • Specific examples of such treatment include a method of agitation at pH 6.0 to 9.0 at a temperature of 40°C to 70°C for 10 minutes to 6 hours.
  • organic solvent treatment and heat treatment can be used in combination.
  • any conditions may be applied, provided that thereby DSA activity is not significantly reduced and L-serin production activity can be reduced or eliminated.
  • Such conditions of organic solvent treatment involve organic solvent concentration that is generally approximately between 20 mM and 2 M, preferably approximately between 20 mM and 1 M, further preferably approximately between 50 mM and 1000 mM, and particularly preferably approximately between 50 mM and 300 mM.
  • organic solvent concentration is generally approximately between 20 mM and 2 M, preferably approximately between 20 mM and 1 M, further preferably approximately between 50 mM and 1000 mM, and particularly preferably approximately between 50 mM and 300 mM.
  • Preferred examples of an organic solvent that is used include: aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde; alcohols such as methanol, ethanol, and isopropyl alcohol; ketones such as acetone; and halogenated hydrocarbons such as dichloroethane.
  • the organic solvent is not limited thereto unless it causes a significant decrease in DSA activity.
  • formaldehyde is the most preferable because it is a substrate for enzyme reactions. Since the addition of D-serine results in production of formaldehyde during such enzyme reaction, a method for adding D-serine instead of formaldehyde may be implemented. It is desired that the concentration of D-serine added be approximately 100 mM to 5 M.
  • the temperature for organic solvent treatment be between 10°C and 50°C and that the pH for the treatment be between 6.0 and 9.0.
  • agitation is desirably carried out such that the pH and the organic solvent concentration become uniform in a treatment solution.
  • enzyme activity may be further stabilized by adding a compound having divalent metal ions, reductants such as 2-mercaptoethanol, dithiothreitol, and sodium bisulfite, and coenzymes such as pyridoxal phosphate and an ammonium salt.
  • reductants such as 2-mercaptoethanol, dithiothreitol, and sodium bisulfite
  • coenzymes such as pyridoxal phosphate and an ammonium salt.
  • Examples of a compound having divalent metal ions include magnesium chloride, manganese chloride, cobalt acetate, ferrous sulfate, and calcium chloride.
  • a concentration of such compound in a treatment solution is generally 0.1 mM to 100 mM and preferably 0.1 mM to 10 mM.
  • L-serine-deaminase-producing bacteria it is also possible to improve optical purity of D-serine as a final product by adding L-serine-deaminase-producing bacteria to a reaction solution after the termination of a D-serine synthesis reaction so as to degrade the L-serine produced.
  • L-serine deaminase-producing bacteria microorganisms lacking D-serine deaminase are desirably used.
  • D-serine can be collected from a reaction solution in accordance with methods that are used in general organic synthetic chemistry, such as extraction using organic solvents, crystallization, thin-layer chromatography, and high-performance liquid chromatography.
  • D-serine can be produced from glycine and formaldehyde with a better yield than is possible with methods for producing D-serine using known DTAs.
  • D-serine, L-serine, and glycine were quantified by high-performance liquid chromatography. Conditions for analyzing them and a method for measuring activities of enzymes (DSA and DTA) were as follows.
  • Detection was carried out by a post-column derivatization method ( J. Chromatogr., 83, 353-355 (1973 )) using orthophthalaldehyde (OPA).
  • OPA orthophthalaldehyde
  • An enzyme solution that had been obtained by allowing a cell suspension to be subjected to ultrasonic disruption was adequately diluted.
  • the diluted enzyme solution (0.1 mL) was added to 0.9 mL of 200 mM Tris-hydrochloric acid buffer (pH 8.0) containing 100 mM glycine, 0.1 mM pyridoxal phosphate, 10 mM magnesium chloride, and 5 mM formaldehyde.
  • the resulting solution was subjected to reaction at 30°C for 15 minutes.
  • D-serine produced was analyzed by HPLC so as to measure the activity.
  • 1 unit of such activity was determined to be the capacity for producing 1 ⁇ mol of D-serine per 1 minute.
  • An LB medium (50 ml) was inoculated with Achromobacter xylosoxidans (ATCC9220), Achromobacter denitrificans (NBRC15125), and Achromobacter xylosoxidans (NBRC13495). After overnight culture at 30°C, harvest was carried out, followed by bacteriolysis using a lytic solution containing lysozyme (1 mg/ml). The resulting lysate was subjected to phenol treatment. Then, DNA was allowed to precipitate by ethanol precipitation in accordance with a usual method. The resulting DNA precipitate was recovered by spooling it onto a glass rod and washed so as to be used for PCR.
  • Primers used for PCR were oligonucleotides (obtained by custom synthesis from Hokkaido System Science Co., Ltd.) having the nucleotide sequences set forth in SEQ ID NOS: 1 and 2, respectively, which were designed based on known DTA genes. These primers had Kpn I and Hind III restriction enzyme recognition sequences near the 5' and 3' ends, respectively.
  • PCR was carried out under the following conditions: denaturation at 96°C for 1 minute, annealing at 55°C for 30 seconds, and elongation reaction at 68°C for 1 minute and 15 seconds for 35 reaction cycles.
  • the PCR reaction product and plasmid pUC18 were digested with Kpn I and Hind III , followed by ligation using Ligation High (TOYOBO). Thereafter, the obtained recombinant plasmid was used for transformation of Escherichia coli DH5 ⁇ .
  • the transformed cell line was cultured in an LB agar medium containing 50 ⁇ g/ml of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside).
  • Am-resistant transformed cell line that was formed into a white colony was obtained.
  • a plasmid was extracted from the thus obtained transformed cell line.
  • the nucleotide sequence of the DNA fragment that had been introduced into the plasmid was confirmed in accordance with a usual method for base sequencing.
  • the obtained plasmid having DNA encoding DSA derived from Achromobacter xylosoxidans was designated as pAcDTA1.
  • the plasmid having DNA encoding DSA derived from Achromobacter xylosoxidans was designated as pAcDTA2.
  • the plasmid having DNA encoding DSA derived from Achromobacter denitrificans was designated as pAcDTA3.
  • Escherichia coli DH5 ⁇ was transformed by a usual method using pAcDTA1, pAcDTA2, and pAcDTA3.
  • the obtained transformants were designated as MT-11015, MT-11016, and MT-11017, respectively.
  • a LB medium (100 mL) containing 50 ⁇ g/ml of Am was inoculated with recombinant microorganisms of each transformant after being placed in a 500-mL baffled Erlenmeyer flask. This was followed by culture at 30°C until OD660 reached 0.6. Then, IPTG (isopropyl- ⁇ -thiogalactopyranoside) was added thereto such that the medium contained 1 mM IPTG. This was followed by further shake culture for 16 hours. The culture solution was centrifuged at 13000 rpm for 10 minutes. The obtained cell bodies were suspended in 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing 5 mL of 1 mM magnesium chloride, followed by cryopreservation at -20°C.
  • Tris-hydrochloric acid buffer pH 8.0
  • Fig. 1 shows the results of SDS-polyacrylamide gel electrophoresis analysis of the transformant disruption solution.
  • the activity fraction was allowed to adsorb hydrophobic chromatography resin (HiTrap Phenyl FF produced by Amersham), followed by linear-gradient elution from 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing 10 mM magnesium chloride, which had been saturated with ammonium sulfate, to 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing 10 mM magnesium chloride. Note that the above operations were carried out at approximately 10°C.
  • Fig. 2 shows the results of SDS-polyacrylamide gel electrophoresis analysis of a solution subjected to ultrasonic disruption, an activity fraction subjected to ion-exchange chromatography treatment, and an activity fraction subjected to hydrophobic chromatography treatment.
  • the molecular weight of purified DSA monomer was 40000 ⁇ 5000.
  • the purified DSA enzyme solution (150 units) was added to a substrate solution comprising 100 mM formaldehyde, 100 mM glycine, 0.1 mM PLP, 10 mM magnesium chloride, and 100 mL of 200 mM Tris-hydrochloric acid buffer (pH 8.0). The resultant was subjected to reaction at 30°C for 20 hours.
  • the enzyme solution subjected to ultrasonic disruption (150 units) of the cell line MT-11015 produced in Example 1 was added to a substrate solution comprising 100 mM formaldehyde or acetaldehyde as an aldehyde source, 100 mM glycine, 0.1 mM PLP, 10 mM magnesium chloride, and 100 mL of 200 mM Tris-hydrochloric acid buffer (pH 8.0).
  • the resultant was subjected to reaction at 30°C for 20 hours.
  • the enzyme solutions (150 units each) subjected to ultrasonic disruption of the recombinant microorganisms produced in Example 1 were separately added to a substrate solution comprising 100 mL of 200 mM Tris-hydrochloric acid buffer (pH 8.0) containing 100 mM formaldehyde, 100 mM glycine, 0.1 mM PLP, and 10 mM magnesium chloride.
  • the resultants were subjected to reaction at 30°C for 20 hours.
  • Table 1 shows the results.
  • the entire nucleotide sequence (GenBanak accession number: U00096) of genomic DNA of Escherichia coli is known to the public. Also, the amino acid sequence of Escherichia coli D-serine deaminase and the nucleotide sequence (GenBank accession number: J01603) of the gene thereof (hereafter to be abbreviated in some cases as dsdA) have already been reported.
  • PCR was carried out using genomic DNA of Escherichia coli cell line W3110 (ATCC27325) as a template and oligonucleotides having the nucleotide sequences set forth in SEQ ID NOS: 9, 10, 11, and 12, which had been produced based on genetic information regarding a region in the vicinity of dsdA of genomic DNA of Escherichia coli cell line W3110.
  • the obtained DNA fragments were digested with Pst I and Xba I and with Xba I and Kpn I , respectively, which are restriction enzymes. Thus, approximately 900-bp and 800-bp fragments of each DNA fragment were obtained.
  • the resulting DNA fragments were mixed with fragments obtained by digesting a temperature-sensitive cloning vector pTH18cs1 (GenBank accession number: AB019610) ( Hashimoto-Gotoh, T., Gene, 241, 185-191 (2000 )) with Pst I and Kpn I , followed by ligation using a ligase.
  • the resultant was transformed into a DH5 ⁇ cell line at 30°C.
  • a transformant that was able to grow on an LB agar plate containing 10 ⁇ g/ml of chloramphenicol was obtained.
  • the obtained colony was cultured overnight at 30°C in an LB liquid medium containing 10 ⁇ g/ml of chloramphenicol so that a plasmid was recovered from the obtained cell bodies.
  • the obtained plasmid was digested with Xba I so as to be subjected to blunt-end treatment with T4DNA polymerase. Thereafter, the plasmid was ligated with a kanamycin-resistant gene derived from pUC4K plasmid (Pharmacia).
  • the thus obtained plasmid was transformed into Escherichia coli cell line W3110 (ATCC27325) at 30°C, followed by overnight culture at 30°C on an LB agar plate containing 10 ⁇ g/ml of chloramphenicol and 50 ⁇ g/ml of kanamycin.
  • a transformant was obtained.
  • An LB liquid medium containing 50 ⁇ g/ml of kanamycin was inoculated with the obtained transformant, followed by overnight culture at 30°C.
  • the resultant was applied to an LB agar plate containing 50 ⁇ g/ml of kanamycin so as to obtain the culture cell bodies. Thus, colonies that were able to grow at 42°C were obtained.
  • the obtained colonies were cultured overnight at 30°C in an LB liquid medium containing 50 ⁇ g/ml of kanamycin.
  • the resultant was further applied to an LB agar plate containing 50 ⁇ g/ml of kanamycin so as to obtain colonies that were able to grow at 42°C.
  • the obtained cell line was designated as a W3110dsdA-deficient cell line (hereafter to be abbreviated in some cases as ⁇ dsdA). Transformation was carried out using the plasmids produced in Example 1 so that cryopreservated cell bodies were produced as described above. Then, a similar reaction was carried out. As a result, substantially no D-serine degradation was confirmed.
  • Formaldehyde was added to a lysate of the frozen cell bodies of MT-11015 produced in Example 1 such that the lysate contained 100 mM formaldehyde, followed by mild agitation at 30°C for 1 hour. During agitation, the pH was adjusted to 8.0 with sodium hydroxide. The resulting suspension of the cell bodies was subjected to the same reaction as that of Example 8. When formaldehyde treatment was not carried out, 2 mol% of L-serine was produced. When formaldehyde treatment was carried out, it was impossible to detect L-serine.
  • An LB medium 50 ml was inoculated with Xanthomonas oryzae (IAM1657), which is obtainable from the Institute of Molecular and Cellular Biosciences at the University of Tokyo. After overnight culture at 30°C, harvest was carried out, followed by bacteriolysis using a lytic solution containing lysozyme (1 mg/ml). The resulting lysate was subjected to phenol treatment. Then, DNA was allowed to precipitate by ethanol precipitation in accordance with a usual method. The resulting DNA precipitate was recovered by spooling it onto a glass rod and washed so as to be used for PCR.
  • IAM1657 Xanthomonas oryzae
  • Primers used for PCR were oligonucleotides (obtained by custom synthesis from Hokkaido System Science Co., Ltd.) having the nucleotide sequences set forth in SEQ ID NOS: 13 and 14, respectively, which were designed based on a known DTA gene of Xanthomonas oryzae (GenBanak accession number: E05055). These primers had Kpn I and Hind III restriction enzyme recognition sequences near the 5' and 3' ends, respectively.
  • PCR was carried out under the following conditions: denaturation at 96°C for 1 minute, annealing at 55°C for 30 seconds, and elongation reaction at 68°C for 1 minute and 15 seconds for 35 reaction cycles.
  • the PCR reaction product and plasmid pUC 18 were digested with Kpn I and Hind III, followed by ligation using Ligation High (TOYOBO). Thereafter, the obtained recombinant plasmid was used for transformation of Escherichia coli DH5 ⁇ .
  • the transformed cell line was cultured in an LB agar medium containing 50 ⁇ g/ml of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside). Thus, an Am-resistant transformed cell line that was formed into a white colony was obtained. A plasmid was extracted from the thus obtained transformed cell line.
  • the nucleotide sequence of the DNA fragment that had been introduced into the plasmid was confirmed to be a sequence identical to the sequence of DTA of Xanthomonas oryzae in accordance with a usual method for base sequencing.
  • the obtained expression plasmid was designated as pXDTA1.
  • Escherichia coli W3110 ⁇ dsdA was transformed by a usual method using pXDTA1.
  • the obtained transformed cell line was designated as MT-11028.
  • Escherichia coli W3110 ⁇ dsdA was transformed by a usual method using pAcDTA1, pAcDTA2, and pAcDTA3 obtained in Example 1.
  • the obtained transformants were designated as MT-11015W, MT-11016W, and MT-11017W, respectively.
  • a LB medium (100 mL) containing 50 ⁇ g/ml of Am was inoculated with recombinant Escherichia coli (MT-11015W, MT-11016W, MT-11017W, and MT-11028) after being placed in a 500-mL baffled Erlenmeyer flask. This was followed by culture at 30°C until OD660 reached 1.0.
  • culture was carried out using BMS10 with capacity of 10 L (produced by ABLE). Operations were carried out under the following culture conditions: agitation: 700 rpm; temperature: 30°C; pH (maintained with NH 3 ): 7.2; aeration: 1 vvm; capacity: 5 L; and culture time: 48 hours.
  • a medium used had a medium composition comprising 7 g of polypeptone (Dainippon Pharma), 0.09 g of ferrous sulfate heptahydrate, 1.5 g of ammonium sulfate, 2 g of magnesium sulfate hexahydrate, 2 g of monopotassium hydrogen phosphate, 2 g of dipotassium hydrogen phosphate, and 0.6 g of ADEKA NOL LG126 (Asahi Denka Kogyo K.K.) with respect to 1 L of water, unless specified.
  • glucose was added, resulting in a glucose concentration of 20 g/L. Then, 50 mL of the culture solution in the aforementioned baffled Erlenmeyer flask was used for inoculation. After the glucose that had been added first became depleted under the aforementioned conditions, glucose was supplied at a variable rate (that resulted in less than 0.1 g/L of glucose) during the remaining time such that the total amount of glucose added was 200. g. Cell bodies were collected from the culture solution via centrifugation so as to be frozen at -20°C.
  • Magnesium chloride hexahydrate (1.0 g) was added to 60 g of frozen cell bodies of MT-11028 (with a solid content percentage of approximately 10%). A variety of organic solvents were added thereto in a manner such that the resultant contained the solvents at given concentrations, followed by agitation at 35°C for 1 hour.
  • Cell bodies (weighing 0.22 g as dry cell bodies) were taken from the above processed cell solution.
  • the solution (9 g) used for enzyme activity measurement was added thereto, followed by agitation at 35°C for 20 hours. Then, the ratio between L-serine produced and residual D-serine was measured. Table 2 collectively shows the results.
  • Example 10 Magnesium chloride hexahydrate (1.85 g) and 1.2 g of formaldehyde (37% by weight) were added to 83.4 g of wet cell bodies (with a solid content percentage of approximately 10%) obtained in Example 10. Water was added thereto such that the formaldehyde concentration was adjusted to 0.5%, followed by agitation at 35°C for 1 hour.
  • Glycine (80 g) and 4 g of magnesium chloride hexahydrate (35% by weight) were added to 280 g of water so as to be dissolved therein.
  • Formaldehyde was added thereto such that the concentrations listed below were achieved, followed by control of pH at 7.5 with the use of sodium hydroxide.
  • 30 g of frozen cell bodies obtained in Example 10 were added thereto so as to initiate reaction.
  • formaldehyde was added such that pH was controlled at 7.3. Table 3 collectively shows the results.
  • the entire nucleotide sequence of Escherichia coli genomic DNA is known to the public (GenBanak accession number: U00096). Also, the amino acid sequence of Escherichia coli serine hydroxymethyltransferase and the nucleotide sequence (GenBank accession number: J01620) of the gene thereof (hereafter to be abbreviated in some cases as glyA) have already been reported.
  • PCR was carried out using genomic DNA of Escherichia coli cell line W3110 (ATCC27325) as a template and oligonucleotides having the nucleotide sequences set forth in SEQ ID NOS: 15, 16, 17, and 18, which were produced based on genetic information regarding a region in the vicinity of glyA of genomic DNA of Escherichia coli cell line W3110.
  • the obtained DNA fragments were digested with Bam HI and Pst I and with Pst I and Hind III, respectively, which are restriction enzymes. Thus, approximately 850 bp and 750 bp fragments of each DNA fragment were obtained.
  • the resulting DNA fragments were mixed with fragments obtained by digesting a temperature-sensitive cloning vector pTH18csl (GenBank accession number: AB019610) ( Hashimoto-Gotoh, T., Gene, 241, 185-191 (2000 )) with Bam HI and Hind III , followed by ligation using a ligase.
  • the resultant was transformed into a DH5 ⁇ cell line at 30°C.
  • a transformant that was able to grow on an LB agar plate containing 10 ⁇ g/ml of chloramphenicol was obtained.
  • the obtained colony was cultured overnight at 30°C in an LB liquid medium containing 10 ⁇ g/ml of chloramphenicol so that a plasmid was recovered from the obtained cell bodies.
  • the recovered plasmid was digested with Pst I so as to be subjected to blunt-end treatment with T4DNA polymerase. Thereafter, the plasmid was ligated with a tetracycline-resistant gene derived from transposon Tn10.
  • the thus obtained plasmid was transformed into Escherichia coli W3110dsdA-deficient cell line at 30°C, followed by overnight culture at 30°C on an LB agar plate containing 10 ⁇ g/ml of chloramphenicol and 50 ⁇ g/ml of tetracycline.
  • a transformant was obtained.
  • An LB liquid medium containing 50 ⁇ g/ml of tetracycline was inoculated with the obtained transformant, followed by overnight culture at 30°C.
  • the resultant was applied to an LB agar plate containing 50 ⁇ g/ml of tetracycline so as to obtain the culture cell bodies.
  • colonies that grow at 42°C were obtained.
  • the obtained colonies were cultured overnight in an LB liquid medium containing 50 ⁇ g/ml of tetracycline at 30°C.
  • the resultant was further applied to an LB agar plate containing 50 ⁇ g/ml of tetracycline so as to obtain colonies that grow at 42°C.
  • This Escherichia coli was transformed with plasmid pXDTA1, followed by culture in the same manner as that used in Example 10. Note that 20 mg/L of glycine was added in the case of flask culture and 2 g/L of glycine was added in the case of culture in a fermenter. During culture, glycine used was added at several different times.
  • 300 mM EDTA (3.5 g; pH 7.5) was added to 100 g of frozen cell bodies of MT-11028 obtained in Example 10, followed by agitation at 4°C for 1 hour.
  • the resulting suspension (10 g) was suspended in 10g of 0.5 M potassium phosphate buffer (pH 7.0) containing 20 mM each of manganese chloride, zinc sulfate, cobalt chloride, nickel chloride, calcium chloride, and ferrous chloride, followed by agitation at 4°C for 1 hour.
  • optical purity of D-serine was 96% or more.
  • 50% or more of the activity of the enzyme synthesizing D-serine from formaldehyde and glycine was maintained compared with the activity before organic solvent treatment, even in a case in which a metal salt was used.
  • An LB medium (50 ml) was inoculated with Escherichia coli cell line K-12. After overnight culture at 30°C, harvest was carried out, followed by bacteriolysis using a lytic solution containing lysozyme (1 mg/ml). The resulting lysate was subjected to phenol treatment. Then, DNA was allowed to precipitate by ethanol precipitation in accordance with a usual method. The resulting DNA precipitate was recovered by spooling it onto a glass rod and washed so as to be used for PCR.
  • Primers used for PCR were oligonucleotides (obtained by custom synthesis from Hokkaido System Science Co., Ltd.) having nucleotide sequences set forth in SEQ ID NOS: 19 and 20, respectively, which were designed based on the known L-serine deaminase gene of Escherichia coli (GenBanak accession number: M28695). These primers had Eco RI and Hind III restriction enzyme recognition sequences near the 5' and 3' ends, respectively.
  • PCR was carried out under the following conditions: denaturation at 96°C for 1 minute, annealing at 55°C for 30 seconds, and elongation reaction at 68°C for 1 minute and 30 seconds for 35 reaction cycles.
  • the PCR reaction product and plasmid pUC18 were digested with EcoRI and Hind III , followed by ligation using Ligation High (TOYOBO). Thereafter, the obtained recombinant plasmid was used for transformation of Escherichia coli DH5 ⁇ .
  • the transformed cell line was cultured in an LB agar medium containing 50 ⁇ g/ml of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside). Thus, an Am-resistant transformed cell line that was formed into a white colony was obtained. A plasmid was extracted from the thus obtained transformed cell line.
  • the nucleotide sequence of the DNA fragment that had been introduced into the plasmid was confirmed to be identical to the sequence of a known Escherichia coli L-serine deaminase.
  • the obtained expression plasmid was designated as pSDA1.
  • the Escherichia coli W3110dsdA/glyA-deficient cell line was transformed by a usual method using pSDAl.
  • the obtained transformant was cultured in a fermenter in the same manner as that used in Example 13.
  • reaction was carried out by adding 10 g of the aforementioned cell bodies to a reaction solution.
  • the reaction solution was analyzed by HPLC. Thus, optical purity of D-serine in the reaction solution was found to be 99.9%.
  • the present invention is useful as a method for producing D-serine from glycine and formaldehyde.
  • D-serine obtained by the production method of the present invention is useful, for example, as a medicine intermediate of a starting material of D-cycloserine that is useful as an antituberculous agent.

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